Methods and compositions for treating autoimmune disorders by targeting Kv1.3 ion channels with functionalized lipid-derived nanovesicles

ABSTRACT

Synthesis and pharmaceutical compositions of antibody-functionalized nanovesicles encapsulating ion channel knockout siRNA, and methods of treating autoimmune diseases associated with increased expression and/or hyperactivity of T cells by selectively targeting memory T cells with the nanoparticles, which deliver their siRNA cargo into the cytosol of the TM cell thus reducing ion channel expression and decreasing Ca2+ influx.

PRIORITY CLAIM

This application is a divisional of U.S. application Ser. No.14/863,525, filed Sep. 24, 2015, and claims priority under 35 U.S.C. §119(e) to U.S. Provisional application No. 62/054,491 filed Sep. 24,2014, the entire disclosures of which are incorporated herein by thisreference.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Contract No.1R21AR060966 awarded by the National Institute of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The presently disclosed subject matter relates to pharmaceuticalcompositions and methods effective for treatment of autoimmunedisorders.

BACKGROUND

Appropriate functioning of the immune system is necessary to identifyand eliminate pathogens and malfunctioning/cancerous cells. However,recognition of various proteins, small DNA-sequences or other moleculesproduced by the host body (termed self) as possible pathogenic agentsleads to the onset of chronic diseases of the immune system namedautoimmune diseases. The immune system is comprised of a variety of Tcell subsets, which are responsible for the acquired immune defense.Naïve T cells are those that have never encountered an antigen, whilecentral memory (TC_(M)) and effector memory (TE_(M)) cells werepreviously exposed to a specific antigen, and provide the memoryresponse. TE_(M) are capable of delivering immediate local tissueresponses to antigens on the basis of their reduced activationrequirements and increased frequency. In contrast, TC_(M) cells (whichconstitute ca. 5% of the total memory pool) are capable of rapidlygenerating a large number of effector cells based on their highproliferative capacity and ability to differentiate into effectors.

The pathology of several autoimmune disorders (such as MultipleSclerosis (MS), Type 1 Diabetes Mellitus (T1DM), Rheumatoid Arthritis(RA) and Systemic Lupus Erythematosus (SLE)) has been coupled to thepresence of TE_(M) cells which, in the case of MS and RA, have beenreported to infiltrate the target tissues and contribute to local tissuedamage. For example, in SLE, TE_(M)'s are highly expressed andhyperactive, and are thought to contribute to the cardiovascularcomplications of the disease. Consequently, a therapeutic interventionsuppressing the function of TE_(M) may be beneficial in autoimmunity.

The activation and the subsequent effector functions of T cells, such asproliferation and cytokine release, are firmly linked to the sustainedelevation of intracellular Ca²⁺ concentration ([Ca²⁺]i) triggered by theencounter with an antigen. Ca²⁺ influx induced by antigen presentationoccurs through CRAC (Calcium Release Activated Ca²⁺) channels that workin concert with other ion channels, transporters and pumps.Particularly, to sustain the driving force for Ca²⁺ ions through CRAC,two potassium channels, the voltage-gated Kv1.3 and the intracellularCa²⁺ activated KCa3.1, maintain the negative transmembrane potential. Itwas reported that these two K⁺ channels are differentially expressed inT cell subsets. TE_(M)'s from patients with autoimmune diseases (RA,T1DM, MS) are characterized by the high level of Kv1.3 as compared toKCa3.1 channels, hence, the former dominantly regulates the TE_(M)cells' membrane potential. Indeed, Ca²⁺-dependent activation in thesecells can be prevented by application of specific Kv1.3 blockers. Thepresent investigators previously demonstrated that inhibition of Kv1.3channels with a potent specific inhibitor (ShK from Stichodactylahelianthus, sea anemone) can hamper Ca²⁺-signaling in SLE T cells.

The treatment of autoimmune diseases requires a very careful strategy,as the systemic application of various drugs can inhibit the function ofcells other than the targeted immune cells, which results in ensembleimmunosuppression. Several studies reported that blocking of the Kv1.3channel function by specific peptide toxins and small-molecules inanimal models in vivo can be used to inhibit effector functions as wellas migration of TE_(M) cells in induced autoimmune deficiencies.However, other cell types express Kv1.3 channels (macrophages, dendriticcell, adipose cells, olfactory neurons), thus raising the possibility ofundesirable side effects. Over the past few years more and more papershave been published reporting cell-specific approaches using NPs thathad demonstrated fewer or no side-effects as compared to the systemicapplication of drugs generally.

An exemplary autoimmune disorder is Systemic Lupus Erythematosus (SLE).Currently approved therapies have serious side effects and, in manycases, limited efficacy. Emerging new therapies still undergoingclinical trials focus on the regulation of T and B cell function (Paz,Z., and G. C. Tsokos. 2013, Curr Opin Rheumatol 25:297-303, thedisclosure of which is incorporated herein by this reference). Thesecells, in fact, play an important role in the pathogenesis of SLE. Inparticular, autoantigen-specific memory T (T_(M)) cells infiltrate thetissues, secrete inflammatory cytokines and reactivate accumulating Bcells through cytokine production and direct CD40L-CD40 binding.

The CD40-CD40L interaction plays a particularly important role in SLEpatients because CD40L is overexpressed in these patients' T cells.CD40L is a member of the tumor necrosis factor (TNF) superfamily locatedon activated T cells, and binds its receptor, CD40, on the B cells. Thisinteraction stimulates B cell activation which, in turn, leads toinflammatory cytokine release, autoantibody formation and end-stageorgan damage. Importantly, T_(M) cells guarantee life-long persistenceof immune memory and long-lived active B cells. Therefore, it is widelyaccepted that lupus cannot be cured without disarming these cells.Therapeutic interventions aiming to interrupt the reciprocal interactionbetween B and T cells via the CD40-CD40L pathway have shown someefficacy in SLE patients; however an increased risk of thromboticcomplications have unfortunately halted clinical trials (CD40L isexpressed in platelets). Clearly, new ways of selectively targetingT_(M) cells to disrupt the T-B communication pathways involved in lupusand other autoimmune disorders having similar etiological considerationsare needed.

SUMMARY

Accordingly, the present investigators engineered functionalized lipidnanovescicles enclosing small interfering RNAs “(siRNA,” short doublestranded RNA molecules that can be used to knock down a specific gene)against ion channels expressed in immune cells. The surface of the lipidnanovesicles are functionalized with antibodies against membraneproteins expressed only in specific immune cell subsets. According tospecific embodiments, lipid nanovesicles (as used herein, nanovesicle,nanoparticle and NP have equivalent scope) enclose siRNAs against Kv1.3channels and are functionalized with a monoclonal antibody whichrecognizes the CD45RO isoform of T_(M) lymphocytes (Kv1.3-NP) (Hajdu,P., A. A. Chimote, T. H. Thompson, Y. Koo, Y. Yun, and L. Conforti,2013, Functionalized liposomes loaded with siRNAs targeting ion channelsin effector memory T cells as a potential therapy for autoimmunity.Biomaterials 34:10249-10257, the entire disclosure of which isincorporated herein by this reference). The NPs are internalized intothe cells, and their siRNA cargo is delivered into the cytosol. As aresult, Kv1.3 channel expression is reduced, which in turn leads todecreased Ca²⁺ influx. Embodiments of the invention offer novelcompositions and methods for the treatment of autoimmune diseases whichare associated to hyperactivity of T cells.

Some embodiments of the invention are directed to pharmaceuticalcompositions comprising antibody-functionalized lipid nanovesicles. Thelipid nanovesicles comprise antibody selective for a membrane proteinunique to a target subset of immune system cells and bound to a surfaceof the lipid nanovesicle; and siRNA effective for inhibiting expressionof an ion channel of the target subset cells upon transfection. ThesiRNA is encapsulated within the lipid nanovesicle.

Other embodiments provide methods of treating a patient suffering from acondition of the immune system characterized by overexpressed and/orhyperexcitable immune system cells. The methods comprise administering acomposition formulated to selectively bind to a target subset of theimmune system cells and transfect it with siRNA. In certain aspects thecomposition comprises antibody-functionalized lipid nanovesicles. Theantibody are selective for a membrane protein unique to the targetsubset of immune system cells and are bound to a surface of the lipidnanovesicle. The lipid nanovesicles are encapsulated with siRNAeffective for inhibiting expression of an ion channel of the targetsubset cells upon transfection.

Methods of manufacturing an agent effective for the treatment of achronic immune system disorder characterized by over-expression and/orhyperexcitability of immune system cells are also provided and comprise:identifying a membrane protein substantially unique to a target subsetof the immune system cells; encapsulating siRNA effective for inhibitingan ion channel of the target subset cell into a lipid nanovesicle; andfunctionalizing a surface of the lipid nanovesicle with an antibody tothe membrane protein.

Still other embodiments are directed to methods of treating a patientsuffering from an immune system disorder characterized by hyperexcitableT_(M) cells. Such methods comprise: selectively suppressing the functionof T_(M)-cells and interrupting a CD40-CD40L pathway between B and Tcells of the immune system. Selectively is defined in this context assubstantially avoiding altering expression of non T_(M)-cells.

These and other embodiments and aspects of the invention we be furtherclarified and understood by reference to the Figures and DetailedDescription herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of signaling pathway in an activatedT-cell.

FIG. 2A. Confocal micrograph images of CD3 cells incubated withCD45RO-NPs (darker), and 2B) labeled with CD45RA antibody (lighter).

FIG. 3. Bar graph demonstrating decrease in Kv1.3 currents in CD3 cellsincubated with Kv1.3-NPs.

FIG. 4. Graphical representation of ion channel dependent Ca²⁺ fluxesfrom CD3 cells incubated with either scr-NP or Kv1.3-NP.

FIG. 5. Drawing illustrating basic synthetic protocol for preparation oflipid nanovesicle (NP) according to certain aspects of the invention.

FIG. 6. Drawing illustrating functionalization of a lipid NP andincorporation of Kv1.3 SiRNA (siKv1.3).

FIG. 7. Results of flow cytometry analysis of CD40L expression in CD3+ Tcells that were resting or activated for 3 h with eitheranti-CD3/anti-CD28 antibodies, TG or PMA/lonomycin, set forthgraphically, showing that T-cell activation increases CD40L levels.

FIG. 8A. Representative image of cells acquired by imaging flowcytometry showing activated cells treated with scr-NPs (top) or 8B)siKv.3-NPs(bottom), and 8C) resting T-cells treated with null-NPs andstained with NFAT (darker) and DAPI (lighter) where the nucleartranslocation of NFAT is indicated by the colocalization of channels inthe merged images.

FIG. 9. Bar graph showing percentage of cells showing nucleartranslocation of NFAT in 3 healthy donors.

FIG. 10. CD40L expression in CD45RO+ activated T cells from 3 healthydonors transduced with NPs. Note: data are normalized to activatednull-NPs. *p<0.05

FIG. 11. Graph showing effect of Kv1.3-NPs in SLE T-cells; CD3 cellswere isolated from a SLE patient and treated with either fluorescentlylabeled Kv1.3-NPs or scr-NPs for 24 h. Ca²⁺ influx was induced by TG for3 h. Resting T-cells (no TG treatment, no intracellular Ca²⁺ influx)transduced with scr-NPs were used as controls (middle/gray), cells werestained with anti-CD40L antibody analyzed by flow cytometry. Only cellsexpressing the fluorescent NPs were gated for analysis.

FIG. 12. Bar graph comparing the SCR and siKv1.3 data derived from graphshown in FIG. 11.

FIG. 13. Graphical depiction of CD40L expression in activated cellstreated with Kv1.3-NPs, scr-NPs and null-NPs and resting cellscontaining scr-NPs, showing that Kv1.3-NPs reduce CD40L expression inSLE T_(M) cells.

FIG. 14. Graphical representation of empirical data showing thatKv1.3-NPs decrease CD40L expression in T_(M) cells derived from SLEpatients. (A) Flow cytometry analysis of CD45RO and CD40L expression inCD3+ cells from an SLE patient treated with siKv.3-NPs, scr-NPs orneither (null-NPs) coated with Alexa488-anti-CD45RO antibody andactivated with TG. Testing T cells with null-NP's were used as controls.

FIG. 15A. Bar graph depiction of average CD40L expression in CD45RO+activated T cells from 3 SLE patients tranduced with NPs with datanormalized to activated null-NPs; 15B) Bar graph depiction of averageCD40L expression in CD45RO− activated T cells from 3 SLE patientstranduced with NPs with data normalized to activated null-NPs.

FIG. 16. Histogram of CD40L expression in activated CD45RO+ CD3+ cellsfrom one SLE patient treated with either scr-NP or siKv1.3-NPs.

FIG. 17. Bar graph depiction of average mean fluorescence intensity(MFI) of CD40L expression in activated CD45RO+ CD3+ cells from 3 SLEpatients transduced with scr-NPs or siKv1.3-NPs with data normalized toMFI of scr-NPs.

FIG. 18. Representation of data demonstrating that Kv1.3-NPs induce lossof CD45RO expression in SLE T cells; Flow cytometry experiments forCD45RO expression in CD3+ cells isolated from one SLE patient incubatedwith null, scr or siKv1.3-NPs and then activated with TG. T_(M)(CD45RO+) and CD45RO− populations were identified by drawing rectanglegates;

FIG. 19. Bar graph depiction of ratio of CD45RO+/CD45RO− in activatedCD3 cells isolated from 3 SLE patients.

FIG. 20. Graphical representation of data demonstrating that Kv1.3-NPsdecrease CD45RO expression in CD4+T_(M) cells and switch the cellphenotype from CD45RA− to CD45RA+ from cytometry experiments for CD45ROand CD45RA expression in CD4+T_(M) cells isolated from a healthy donorand incubated with either scr- or siKv1.3-NPs and then activated withTG.

FIG. 21. Graphical representation of data showing viability for cellsshown in FIG. 20 as measured by flow cytometry using the nuclear dye7-AAD.

FIG. 22A. Series of confocal images of T cells incubated withCellVueRed-containing CD45RO-NPs, wherein the CD45RO antibody on the NPswas labeled with Alexa488 secondary mouse antibody (ALexa488-IgG, green)and from left to right depicting a brightfield image of the cells,CellVueRed signal of NPs, Alexa647 fluorescence of SAV, (ULVs attachedT_(M) cells), Alexa-488 fluorescence of 2nd antibody, and a merger ofthe red, blue and green channels; 22B) series of confocal images ofT-Cells treated with lyophilized (24 hr culturing), CellVueRed-labeledCD45RO-NPs depicting from left to right, a brightfield image of thecells, a CellVueRed signal of ULVs (red); image, an Alexa-647fluorescence image, and a merger of the red and blue channels.

FIG. 23. Graphical representation of data showing typical whole-cellcurrent traces of Kv1.3 channels in an activated T cell treated witheither Scramble-NPs or Kv1.3-NPs. Cells were held at −120 mV anddepolarized to +50 mV for 15 ms; P/5 online leak subtraction wasapplied.

FIG. 24. Reduction of Kv1.3 expression in percentages is shown in Tcells treated with Kv1.3-NPs as compared to cells incubated withScramble-NPs (3 donors, n=18). The reduction was determined for eachdonor and average

percent decrease for three donors is indicated (mean±SEM). The percentdecrease in Kv1.3 current is also shown when T cells were transfectedwith specific Kv1.3 (Kv1.3-siRNA bars) and control siRNAs (3 donors,n=15).

FIG. 25. Sets forth empirical evidence that Kv1.3 silencing withKv1.3-NPs impairs Ca²⁺ response in T_(M) cells by depicting atime-course of TG-induced Ca²⁺ influx through CRAC channels in resting Tcells preincubated (24 h) with Kv1.3-NPs (black line) or Scramble-NPs(gray line). Fluorescence intensity of Indo-1 loaded cells was detectedwith flow cytometer only in Alexa-488 gated subpopulation (NPs werefunctionalized with SAV-Alexa488).

FIG. 26. Bar graph comparing ratio of AUC (area under the curve) andCa²⁺ amplitude (ΔCa²⁺) for Kv1.3-NP and Scramble-NP treated cells (n=3donors).

DETAILED DESCRIPTION

The pathogenesis of many autoimmune disorders is characterized byhyperactive and/or over-expressed T_(M) cells. SLE is a typical andtherefore exemplary such autoimmune disorder. In SLE the T_(M) cellsshow a Ca²⁺-dependent increase in the costimulatory CD40 ligand (CD40L),which binds CD40 on B cells, resulting in B cell activation andautoantibody production. CD40L is widely recognized as a potentialtarget for developing therapies for SLE. Cystolic Ca²⁺ levels increaseduring T cell activation and mediate CD40L expression, and are regulatedby Kv1.3 ion channels. As described in Cahalan, M. D., and K. G. Chandy,2009, Ca²⁺ signaling regulates the transcription factor NF-AT whichdrives both cytokine production and CD40L expression in T cells (“Thefunctional network of ion channels in T lymphocytes,” Immunol Rev231:59-87, the entire disclosure of which is incorporated herein by thisreference.) Ca²⁺ signaling is controlled by ion channels. Inhibition ofKv1.3 channels is therefore a desired outcome of pharmacologicaltherapies.

Although in vivo application of Kv1.3 blockers has been used effectivelyin animal models of autoimmunity, pharmaceutical therapeutics have beenlimited because the expression of Kv1.3 channels in other cell typesoften leads to unexpected and undesirable side effects. Targetedsilencing of the Kv1.3 gene in T_(M)'s could be an alternative approach.Effective and selective in vivo delivery of siRNA, however, remains achallenge in the art. The present investigators therefore designed anovel therapy utilizing nanoparticles as selective siRNA deliveryvehicles. The design takes advantage of the fact that TE_(M)'s arecharacterized by the presence of “O” or “0” isoform of CD45R (CD45ROphosphatase) and lack of CD45RA (isoform “A”) and CCR7− (chemokinereceptor 7) in the cell membrane. Also TC_(M) are CD45RO+, however, theyexpress CCR7 and make up a small fraction of memory T cell population.Naïve T cells, on the other hand, constitute a CD45RA+CCR7+ and CD45RO−subpopulation of the T cell pool.

CD45RO antibody-functionalized NPs encapsulated with siRNAs againstKv1.3 channels and selective to human CD45+, effector memory T cellswere designed, synthesized, and tested for suppression of relevantfunction. Fluorescence confocal microscopy was utilized along withimmucytochemistry to test if the binding and the internalization ofCD45RO antibody labeled NPs, as well as the release offluorophore-tagged siRNA into the cytosol, is specific to the targetT_(M) cells. Furthermore, to monitor the effectiveness ofgene-down-regulation by siRNAs encapsulated into NPs, single-cellelectrophysiology (patch-clamp technique) was utilized to determine theexpression/current of Kv1.3 ion channels in T_(M) cells. To assess thefunctional impact of Kv1.3 gene knock-down on the Ca²⁺⁻ response inT_(M) cells, Indo-1 ratiometric Ca²⁺ measurements were taken using aflow cytometer.

As demonstrated by the Examples below, Kv1.3-NPs selectively targetedT_(M) cells, and not naïve T cells. Kv1.3-NPs were effective in reducingNF-AT activation (nuclear translocation) and CD40L expression in healthyT cells. Furthermore, these Kv1.3-NPs corrected the CD40L overexpressionof SLE T_(M) cells. They delivered the encapsulated siRNAs into thetargeted T cells where they suppressed Kv1.3 channel expression and Ca²⁺influx implicated in the pathology of SLE and other autoimmunedisorders.

One embodiment of the invention is directed to pharmaceuticalcompositions. The compositions comprise antibody-functionalized lipidnanovesicles, wherein the antibody is a binding partner for a membraneprotein unique to a target subset of immune system cells. Thenanovesicles encapsulate siRNA effective for inhibiting expression of anion channel of the target subset of cells upon transfection.

Methods for functionalizing lipid nanoparticles with antibody is knownin the art. For example, guidance is provided by “Application ofpoly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE)block copolymers and their derivatives as nanomaterials in drugdelivery” Int J Nanomedicine. 2012; 7: 4185-4198, the entire disclosureof which is incorporated herein by this reference.

The Kv1.3 channel is a voltage-activated potassium (K+) channel thatshows a fast activation and slow C-type inactivation and recovery. Thechannel is encoded by KCNA3, located in humans on chromosome 1 atposition 111214310-111217655 (RGD ID 1342945). While Kv1.3 channels arefound in all T and B cell subsets in the resting state, their expressionis markedly upregulated in activated effector memory T cells (TEMs) andIg class-switched memory B cells from ˜250 to ˜1500 channels per cell.The two major K+ channels that are expressed in lymphocytes, Kv1.3 andKCa3.1, are promising targets for the treatment of autoimmune disorders,including but not limited to multiple sclerosis, type 1 diabetes,rheumatoid arthritis, psoriasis, systemic lupus erythematosus, andrapidly progressive glomerulonephritis. Moreover, KCa3.1 is related toacute immune responses and Kv1.3 is related to chronic immune responses.Combined inhibition may enhance effects in autoimmune disorders or otherconditions where suppression of the immune system is desired, e.g.graft/organ rejection syndrome. In specific embodiments the siRNA iseffective for inhibiting an ion selected from Kv1.3, KCa3.1 andcombinations thereof. In more specific embodiments the siRNA iseffective for inhibiting Kv1.3. Where a functionalized nanovesicle isencapsulated with siRNA effective for inhibiting Kv1.3, for example, itmay be referred to herein as siKv1.3. Inhibition may be effectuated bycomplete or partial knockout (sometimes referred to as “knockdown”) ofKCNA3 or by knockout/knockdown of any gene necessary for the synthesisor functioning of Kv1.3.

In specific embodiments, the immune system cells are T-cells and thetarget subset of the T-cells is a CD45RO positive isoform of T_(M)cells. An exemplary effective binding partner may be CD45RO antibody orany fragment thereof retaining binding efficacy for CD45RO+. In specificembodiments the antibody comprises monoclonal antibody. In very specificembodiments the T-cell comprises T_(M).

A person of ordinary skill in the art is aware that many different siRNAmay be generated for effective knockdown/knockout of a given gene, andthat generation of siRNA is within the skill of the ordinary artisan.Common methods for generating siRNA effective for knockdown or knockoutof a given gene include chemical synthesis, in vitro transcription,digestion of long dsRNA by an RNase III family enzyme (e.g. Dicer, RNaseIII), expression in cells from an siRNA expression plasmid or viralvector, and expression in cells from a PCR-derived siRNA expressioncassette. Guidance is provided, for example, in Sui G, Soohoo et al.(2002) A DNA vector-based RNAi technology to suppress gene expression inmammalian cells. Proc Natl Acad Sci USA 99: 5515-20, Brummelkamp T R, etal. (2002) A system for stable expression of short interfering RNAs inmammalian cells. Science 296: 550-3, and Paul C P et al. (2002)Effective expression of small interfering RNA in human cells. NatureBiotechnology 20: 505-8, the entire disclosures of which areincorporated herein by this reference.

In specific embodiments the siRNA aspect of the compositions comprises a19-25 nucleotide Kv1.3-specific siRNA. According to very specificembodiments, the siRNA is selected from the group consisting ofGUAACUCGACUCUGAGUAAtt (SEQ ID NO: 1), UUACUCAGAGUCGAGUUACtt (SEQ ID NO:2), CUUACCCUCUCUCUCUUAAtt (SEQ ID NO: 3), UUAAGAGAGAGAGGGUAAGtt (SEQ IDNO: 4), GAUGGACCUUUCAACGUUAtt (SEQ ID NO: 5), UAACGUUGAAAGGUCCAUCtt (SEQID NO: 6), and duplexes thereof.

In some embodiments, the composition is formulated for systemicadministration. According to specific embodiments the composition may beformulated for parenteral administration, for example, intravenous,intra-arterial, intraosseous infusion, intracerebral,intracerebroventricular, and intrathecal administration. Lipidnanovesicles are ideal for delivery via injectable routes since they arebiocompatible and solutions may be formulated surfactant-free.

Other embodiments are directed to methods of treating a patientsuffering from a condition of the immune system characterized byoverexpressed and/or hyperexcitable immune system cells. The methodscomprising administering a composition according to aspects of theinvention, formulated to selectively bind to a target subset of theimmune system cells and transfect it with siRNA effective to knockdownor knockout an ion channel implicated in Ca²⁺ flux. The compositionscomprise: lipid nanovesicles, lipid nanovesicles functionalized withantibody selective for a membrane protein unique to the target subset.In specific embodiments the antibody comprises monoclonal antibody. Theantibody is bound to a surface of the lipid nanovesicle either directlyor indirectly. The nanovesicle is encapsulated with siRNA effective forinhibiting expression of an ion channel of the target subset cells upontransfection. According to specific embodiments, the immune system cellis a T-cell, the subset of the T-cell is a CD45RO+ isoform, the membraneprotein is unique to the CD45RO positive isoform, and the antibodycomprises anti-CD45LRO. In very specific embodiments the T-cellcomprises a T_(M) cell.

In specific method embodiments the ion channel comprises Kv1.3 and thesiRNA comprise one or more of a 19-25 nucleotide Kv1.3-specific siRNA,for example, the siRNA may be selected from the group consisting ofGUAACUCGACUCUGAGUAAtt (SEQ ID NO: 1), UUACUCAGAGUCGAGUUACtt (SEQ ID NO:2), CUUACCCUCUCUCUCUUAAtt (SEQ ID NO: 3), UUAAGAGAGAGAGGGUAAGtt (SEQ IDNO: 4), GAUGGACCUUUCAACGUUAtt (SEQ ID NO: 5), UAACGUUGAAAGGUCCAUCtt (SEQID NO: 6), and duplexes thereof.

Although this disclosure illustrates a broad embodiment of the inventionby specific example (SLE), the compositions and methods are applicableto any autoimmune disorder characterized by hyperexcited orover-expressed T_(M) cells. Exemplary autoimmune disorders which may beeffectively treated include, but are not limited to Rheumatoidarthritis, Type 1 diabetes, Multiple Sclerosis, Psoriasis, Vasculitis,Alopecia areata, Systemic Lupus Erythematosus, Polymyalgia rheumatica,Ankylosing spondylitis, Celiac disease, Sjögren's syndrome, and Temporalarteritis.

According to specific embodiments, a therapeutic regimen comprisesdosing according to a schedule based on severity of the disease across atherapeutic time frame. Severity of the disease may be assessed, forexample, by calculating a ratio of T_(M) to naïve T cells in plasma ofthe patient sampled across the therapeutic time frame. Autoimmunedisorders are generally characterized by disruption in ordinaryhomeostasis and an increase in the ratio. Treatment designed toeffectuate a reduction in T_(M) cells should restore a nonpathologicalequilibrium across the therapeutic time frame.

Embodiments providing methods of manufacturing an agent, specifically asolid lipid nanoparticle/nanovesicle, effective for the treatment of achronic immune system disorder characterized by over-expression and/orhyperexcitability of immune system cells, are also provided. In specificembodiments the methods comprise: identifying a membrane proteinsubstantially unique to a target subset of an immune system cellpopulation; encapsulating siRNA effective for inhibiting an ion channelof the target subset cell into a lipid nanovesicle; and functionalizinga surface of the lipid nanovesicle with an antibody to the membraneprotein.

A fundamental advantage of the instant methods relates to theselectivity for TM cells over naive T cells or other cell populations.Embodiments of the invention provide methods of treating a patientsuffering from an immune system disorder characterized by hyperexcitableT_(M) cells by selectively suppressing the function of T_(M) cells andinterrupting a CD40-CD40L pathway between B and T cells of the immunesystem, wherein selectively is defined as substantially avoidingaltering expression of non T_(M)-cells.

The present subject matter demonstrates for the first time that geneexpression of a T cell subpopulation can be manipulated by lipid-basedunilamellar, antibody-functionalized NPs. The data generated for theillustrative embodiments verifies that PEG-coated lipid NPs can berapidly functionalized with antibodies against specific T cell markersusing streptavidin-biotin complex. The NPs functionalized with CD45ROantibodies selectively bind to a subtype of T cells, the CD45RO+ T_(M)'sthat are key players in chronic immune disorders like autoimmunity. Datafurther demonstrates that T_(M) cells bind and endocytose the NPs loadedwith siRNAs. Ultimately, patch-clamp measurements demonstrate theefficient knock-down of Kv1.3, and flow cytometric Ca²⁺-flux experimentsshow that down-regulation of Kv1.3 channels by Kv1.3-NPs inhibits Ca²⁺influx into T_(M) cells. Since Ca²⁺ influx is one of the earliest eventsin T cell activation, these data indicate that Kv1.3-NPs are effectiveimmune suppressive agents that selectively reduce T_(M) cell activation.It will be apparent to a person of ordinary skill in the art that thespecific inventive nanovesicles disclosed herein may be easily modifiedto target other cells and/or deliver other drugs/siRNAs and may furtherbe developed into personalized therapeutics, i.e. specifically tailoredto individual patients.

The following examples are intended to illustrate particularembodiments, aspects, and features of the invention and should not beconstrued to limit the full scope as defined by the appended claims.

EXAMPLES Example 1

This example demonstrates synthesis of an antibody-functionalized lipidnanovesicle according to specific embodiments of the invention.

Human T lymphocytes were isolated from the blood of healthy consenteddonors and discarded blood units from Hoxworth Blood Center (UC,Cincinnati) using RosetteSep™ Human Total Lymphocyte Enrichment Cocktail(StemCell Technologies). The protocol was approved by University ofCincinnati IRB. T cells were maintained in RPMI-1640 medium supplementedwith 10% human serum, 200 U/ml penicillin, 200 μg/ml streptomycin and 10mM HEPES (T cell medium). Cells were activated with 4-10 μg/ml PHA(phytohemaglutinnin-A, Sigma-Aldrich) in presence of peripheral bloodmononuclear cells (PBMC) for 72 hrs.

NP Preparation

Chloroform-dissolved lipids L-α-phosphatidylcholine (PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (PE-PEG-biotin) and cholesterol (CH) (Avanti Polar LipidsInc.) were mixed in a 3:1:1 mole ratio, dried with N₂ gas, rehydratedwith PBS (pH=7.4), and shaken in an incubator at 37° C. for 2 hours tomake multilamellar vesicles (MLV). After sonication (Misonix, XL-2000series), the sample was extruded with 100 nm filter to synthesizeunilamellar vesicles (ULV=NP) (LIPEX™ Thermobarrel Extruder, NorthernLipids Inc.). NPs with lipid dye CellVueRed (Molecular TargetingTechnologies Inc.) were prepared as described above except the lipid dyewas added to the lipid mixture before drying with N₂.

Functionalization of NPs

Biotynilated antibodies (mouse anti-human IgG and CD45RO, 10 μg/ml, BDBiosciences) were first incubated with 10 μg/ml Alexa-647 or Alexa-488conjugated streptavidin (SAV, Life Technologies) in PBS. Then theantibody-SAV complex was added to the 100 nm NP and incubated at roomtemperature. The unbound antibody and SAV was removed using CL-4Bcolumns (GE Healthcare Life Sciences). NPs later used for siRNAencapsulation were frozen at −80° C. for 2-4 hrs, and then lyophilizedfor 48 hrs (Labconco, FreeZone 6 Freeze Dryer).

siRNA Encapsulation into NPs

Lyophilized CD45RO-NPs (app. 50 μg lipid) were reconstructed in 100 μlnuclease-free water containing 200-400 pmol of either Kv1.3-siRNA(Kv1.3-NPs; Santa-Cruz Biotechnology Inc.) or scramble Cy3-siRNA(Scramble-NPs Applied Biosystems) complexed with protamine-sulfate (1:5molar ratio).

Size Measurement of NPs

Dynamic Light Scattering (DLS) and intensity fluctuation correlationmethods was used to determine NPs diameter with Zetasizer Nano ZS(Malvern Instrument). ULVs were visualized using scanning electronmicroscopy (SEM, Hitachi SU 8000), scanning transmission electronmicroscopy (STEM), and WETSEM for hydrated samples. Briefly, for TEMobservation, lyophilized nanoparticles were first dispersed in Methanoland lipid solution (50 μL) was dropped and dried on Cu grid (TED PELLA,G200HS). The samples were inserted and visualized in the STEM microscopeat 30 kV. Also, lipid NPs were visualized in hydrated state usingWETSEM™ technology (EI-Mul Technology, Israel). Liquid dish membrane (QX102 capsule) was first coated by poly-L-lysine and suspended NPssolution(15 μL) were attached on the membrane and lipid vesicles were visualizedusing SEM at 25 kV.

Immunocytochemistry

T cells incubated with antibody-coated NPs were plated ontopoly-L-lysine coated glass coverslips and fixed with 1% formaldehyde.When cells were labeled with mouse anti-human CD45RA-Alexa488 antibody(Biolegend) to test CD45RO-NPs specificity, blocking with 10% FCS in PBS(pH 7.4) preceded incubation with the CD45RA antibody. Coverslips weremounted onto glass slides using Fluoromount G (eBioScience).

Confocal Microscopy

Zeiss LSM 510 META was used for confocal images of the cells. The He—Nelaser was selected to excite fluorophore Alexa647 (line 633 nm) andCy3/CellVueRed (line 543 nm), and Argon laser (line 488 nm) to visualizeAlexa488. The thickness of the slices and z-stacks were set to 1 μm.

Electrophysiology

Kv1.3 currents were recorded using Axopatch 200B amplifier (MolecularDevices) in whole-cell voltage-clamp configuration. The bath solutionwas (in mM): 145 NaCl, 5 KCl, MgCl₂, 2.5 CaCl₂, 5.5 glucose, 10 HEPES(pH 7.35). The pipette solution contained (in mM): 140 KF, 11 K2EGTA, 1CaCl₂, 2 MgCl₂, and 10 HEPES (pH 7.22) [21]. Kv1.3 currents were evokedby 15-ms-long depolarizations to +50 mV from a holding potential (HP) of−120 mV. The amplitude of the peak current was determined at +50 mV, andthe current density (CD) was given as the ratio of peak current at +50mV and the whole-cell cell capacitance (which is a measurement of cellsize/surface area). The CD is proportional to the number of Kv1.3channels per unit area.

Cell Transfection

T cells were transected by nucleofection with Kv1.3 specific andscramble Cy3-labeled siRNAs along with pmaxGFP using 4D-NucleofectorSystem according to the manufacturer's protocol (Lonza Group Ltd.) Thecells were studied 24 hours post transfection.

Treatment of T Cells with siRNA-Encapsulated NPs (siRNA-NPs)

3×105 T cells (either activated, for electrophysiological experiments,or resting, for Ca²⁺ measurements) in T cell medium were mixed with 50μl of siRNA-NPs, and incubated for 24 h in cell culture incubator (37°C., 5% CO₂, humidified).

Ca²⁺ Measurement

Ca²⁺ was measured using the Ca²⁺ add-back method as described by Baba etal. Briefly, 1×106 CD3+ cells were loaded with 1:1000 fold of 2 mg/mlIndo-1/AM ratiometric dye and 0.015% Pluronic 127 (Life Technologies,Carlsbad, Calif.) in Hank's balanced salt solution containing 1 mMCaCl₂, 1 mM MgCl₂ and 1% FCS for 30 min at 37° C., then washed threetimes in Hank's balanced salt solution supplemented with 10 mM HEPES (pH7.0) and 1% FCS. Prior to measurements, cells were resuspended in acalcium-depleted solution prepared from the Hank's balanced saltsolution/HEPES solution mentioned above and supplemented with 0.5 mMEGTA (pH 7.4). Samples were kept at 37° C. until analysis. Indo-1fluorescence ratio (indicative of the [Ca²⁺]i) in T cells were measuredby flow cytometry on an LSRII flow cytometer (Beckton Dickinson) using a20 mW UV (355 nm) laser and capturing fluorescence using 505 nm longpass and 530/30 band pass filters for unbound Indo-1 and a 405/20 bandpass filter for Ca²⁺-bound Indo-1. Changes in Indo-1 fluorescence ratiowere measured in T_(M) cells by gating on CD45RO-NPs labeled withSAV-Alexa488.

The following protocol was implemented. Cells were exposed tothapsigargin (TG, 1 μM) in 0 mM Ca²⁺ solution followed by the 2 mMCa²⁺-containing solution. This protocol allows measuring Ca²⁺ influx,which originates exclusively through CRAC channels. Exposure of thecells to TG leads to depletion of intracellular Ca²⁺ stores andactivation of the signaling steps necessary for opening of CRACchannels. Ca2+ influx through the opened CRAC channels is then inducedby increasing the extracellular Ca²⁺ concentration ([Ca²⁺]). Sampleswere recorded at 300 events/second on a ‘low’ flow rate. Analysis of thekinetics was performed using Flow-Jo software (Tree Star Inc).

Area under the curve (AUC) was calculated for that part of Ca²⁺-responsecurve when cells were bathed in 2 mM Ca²⁺ solution after TG addition,and it estimates the average Ca²⁺ influx into the cell. Ca²⁺ amplitudes(ΔCa²) was given as the peak intensity ratio of Indo-1 upon addition of2 mM Ca2+ corrected with the mean Indo-1 ratio at 0 mM Ca2+ before TGaddition (baseline was subtracted from the peak value).

All reagents were purchased from Sigma-Aldrich Ltd., if not otherwisestated. ShK was bought from Bachem Holding AG. Statistical comparisonwas performed using Student's t-test; the significance level was set to0.05. The values are given as mean±SEM.

Results

CD45RO-NPs specifically bind to and are internalized by T_(M) cells.

In cell-targeted therapy it is critical to design NPs which can attachwith their cargo only to the selected cells/tissues. Hence, whetherCD45RO-NPs were able to bind selectively to T_(M) cells was confirmed bythe following procedure. Primary human T cells were exposed tofluorescent (SAV conjugated with Alexa647, blue)lyophilized/reconstructed CD45RO-NPs or IgG-NPs. After 24 hr incubation(at 37° C. in the presence of 10% human serum), the cells were fixed,and naïve T cells were labeled with Alexa488 conjugated CD45RA antibody(FIG. 2B). Confocal micrographs demonstrated that CD45RO-NPs bound onlyto T_(M) but not naïve T cells. The merged images showed clearly thatthe cells, which were decorated with CD45RO-NPs, do not express CD45RA.To show the lack of CD45RO-NP non-specific binding, T cells wereincubated with IgG-NPs. Confocal images illustrated that IgG-NPs did notadhere to the membrane of T cells.

CD45RO-NPs Specifically Bind to CD45RO+ T_(M) Cells

Further experiments showed that bound CD45RO-NPs were internalized byT_(M) cells (FIG. 6). CD45RO-NPs labeled with CellVueRed lipid dye andSAV-Alexa647 were utilized. After a 24-hour-incubation of T cells withthese NPs, cells were plated and the CD45RO antibodies on the NPs, whichhad already interacted with the cells, were labeled with Alexa488secondary antibody. Since this latter step occurs in non-permeabilizedcells, this intervention allows exclusive staining of the CD45RO-NPsattached to the cell membrane. Therefore, the merge image demonstratedinternalization of NPs by purple fluorescence, as well as their fusionto the plasma membrane (white and yellow fluorescence) Similardistribution was observed with CD45RO-NPs that underwent lyophilizationindicating that this procedure did not compromise the antibody binding,i.e. the antibodies remained attached to the surface of the NPs. Asshown before, the fluorescence signals of SAV-Alexa647 and CellVueRed ofthe NPs were co-localized only in a subset of T cells. Overall theseexperimental results confirm that lyophilized CD45RO-NPs can selectivelytarget and accumulate in/on T_(M) cells. Hence, CD45RO-NPs represent asuitable carrier for siRNA delivery and gene knock-down in T_(M)'s.

T_(M) Cells Endocytose CD45RO-NPs; Kv1.3 siRNA Delivery to T_(M) Cells

Even though the CD45RO-NPs recognize, bind to and enter the T_(M)'s,this does not automatically mean that the encapsulated cargo is releasedinto the cytosol. Consequently, in the next step CD45RO-NPs loaded witha Cy3-labeled scramble siRNA to visualize whether these NPs are able todeliver siRNAs into T_(M) cells were utilized. As detailed above,freeze-dried CD45RO-NPs were reconstructed in protamine-complexedcontrol Cy3-siRNA containing water and added to primary T cell for 24hrs. CD45RO-NPs (SAV-Alexa647, blue) loaded with Cy3-siRNA (red) werecapable of attaching to TM cells selectively, and the siRNAs weredetected intracellularly in CD45RO+ cells that picked up the CD45RO-NPs(merged image). A further confirmation of specific NP-assisted siRNAdelivery was shown in confocal snapshots of T cells treated with nakedCy3-siRNA/protamine solution for 24 hr. No intracellular redfluorescence of Cy3-siRNA was detected. Overall, these findings showthat CD45RO-NPs are able to serve as transporters of siRNAs into theT_(M)'s.

Cy3-siRNA Uptake of T_(M) cells; Kv1.3-NPs Reduce Expression of Kv1.3Channels

To measure the efficacy of Kv1.3 channels' knock-down by Kv1.3-NPs, asingle cell technique—patch-clamping—was applied to compare the currentsthrough Kv1.3 channels in Kv1.3-NPs (Kv1.3 siRNA loaded CD45RO-NPs) andScramble-NPs (control siRNA encapsulated CD45RO-NPs) treated cells.These measurements allow estimation of the expression of functionalKv1.3 channels in single TM cells. In these experiments, the Alexa488fluorophore conjugated SAV moiety of the NPs was used to visualize thecells that have bound/incorporated the NPs. Only cells that displayedgreen fluorescence were selected for electrophysiological recording. Theknock-down efficiency of Kv1.3-NPs was compared with that of naked (nonNP encapsulated/transfected; see materials and methods) Kv1.3 siRNAs.These Kv1.3 siRNAs were shown to be effective inhibitors of Kv1.3expression. Activated T cells were co-transfected with naked Kv1.3 orscramble siRNAs and a GFP-encoding plasmid (moles of siRNA far exceededthat of the pMaxGFP vector). GFP-expressing cells were selected toundergo patch-clamping as they also contained siRNAs. Whole-cell Kv1.3current traces in T cells treated with Scramble-NPs and Kv1.3-NPs wererecorded. The Kv1.3 current was significantly lower in the cells treatedwith Kv1.3-NPs than in Scramble-NPs' treated cells. The extent of thedecrease in Kv1.3 peak current and CD was app. 60% (3 donors for eachexperiment, n≥15 cells) in Kv1.3-NP treated cells and Kv1.3-siRNAtransfected cells. These data prove that Kv1.3 siRNAs delivered byCD45RO-NPs effectively down-regulated the expression of Kv1.3 channelsin T_(M) cells.

CD45RO-Kv1.3NPs Down-Regulate Kv1.3 Expression in T_(M) Cells;Ca²⁺-Signaling in Memory T Cells was Reduced by Kv1.3-NP Gene Knock-Down

Kv1.3 channels play an important role in the regulation of Ca²⁺signaling in T_(M) cells, and the inhibition of these channels reducesthe influx of Ca²⁺ through the CRAC channels. Hence, the Ca²⁺-responseof T cells treated with siRNA-loaded CD45RO-NPs was studied. In this setof experiments, resting T cells were incubated with either Kv1.3-NPs orScramble-NPs. Cells treated with empty CD45RO-NPs or nothing served asfurther controls. Twenty-four hours after treatment, Ca²⁺ measurementswere performed. These were performed by flow cytometry, which allowsgating on T_(M) cells. Indol-loaded T cells were kept in Ca²⁺-freeextracellular solution. Addition of TG (1 μM) induced a small increasein the [Ca²⁺]i (measured as an increase in the fluorescence ratio ofIndo-1), which corresponds to the emptying of intracellular Ca²⁺ store(ER, endoplasmic reticulum). This intervention is used to artificiallyopen the CRAC channels because it activates a Ca²⁺ sensor in the ER,which moves in proximity and opens the pore forming subunit of the CRACchannel in the plasma membrane. Yet, no further [Ca²⁺]i increase isobserved because the extracellular solution contains no Ca²⁺. At thispoint the external solution was changed to one containing 2 mM Ca²⁺.Immediately upon increasing the extracellular Ca²⁺, Ca²⁺ influx throughCRAC channels can be detected. This is depicted by the robust increasein [Ca²⁺]i, which is monitored via the increase of the fluorescenceratio of Indo-1 at 400 and 475 nm. To validate flow-cytometric Ca²⁺measurements and confirm the role of Kv1.3 in Ca²⁺-response 10 nM ShK, aKv1.3 antagonist, was applied. Silencing the Kv1.3 gene by Kv1.3-NPsresulted in a remarkable decrease in Ca²⁺ influx as compared toScramble-NPs' treated cells. The effect of Kv1.3-NPs on Ca²⁺ uptakethrough CRAC channels was quantified by calculating the ratio of thearea under curve (AUC) and Ca²⁺ peak amplitudes (ΔCa2+) forKv1.3-silenced and control cells. Both parameters of Ca²⁺ signalingdecreased significantly upon Kv1.3-NP treatment of T_(M)'s (3 donors).Comparable baseline and peak [Ca²⁺]i in untreated cells, and cellstreated with empty CD45RO-NPs and Scramble-NPs are indicative of a lackof effect of CD45RO antibody on Ca²⁺ fluxes. These data indicate thatKv1.3 silencing with Kv1.3-NPs impairs Ca²⁺ response in T_(M) cells andKv1.3-NPs are an effective alternative to Kv1.3 pharmacologicalblockers.

The foregoing illustrates successful synthesis and specific targeting ofT_(M) cells by anti-CD45RO-NPs. Successful transfection is highlydependent on the freeze-drying procedure involved in fabrication, neededto include siRNA into the NPs, during which the detachment offunctionalizing antibodies can occur due to the non-covalent,biotin-streptavidin linkage. This could give rise to the non-selectiveattachment of NPs to naïve T cells (bare NPs) or simply binding ofCD45RO-fluorophore-conjugated-SAV complex to the T_(M)'s. In the imagesset forth in FIGS. 22A and 22B, only SAV-fluorophore “positive” T cellsare coated by CellVueRed-containing, CD45RO-NPs. These lipid dye labeledCD45RO-NPs can be stained with secondary antibodies when they are linkedto the cell surface of TM's. Thus, stable and effective NPs wereproduced. The efficiency of the CD45RO-NPs was confirmed by confocalmicrographs showing the cytosolic accumulation of Cy3-conjugated controlsiRNAs (not shown) and functional studies (FIGS. 23, 24 and 25).Patch-clamp experiments verified that Kv1.3-NPs were able to suppressKv1.3 current/expression in TM cells. The 60% knock-down efficiency is aresult of the efficacy of the siRNAs and not the amount of siRNAs in theNPs taken up by the cells since transfection of T cells with naked,specific siRNAs also had the same reduction in the Kv1.3 peak current.Further flow-cytometric Ca²⁺ measurements also underline thatKv1.3-siRNA was effectively delivered into the cells by CD45RO-NPs andcould reduce the number of Kv1.3 channels in TM cells: theKv1.3-regulated Ca²⁺ influx of T cells was significantly decreased (FIG.26).

Example 2

This example illustrates application of embodiments of the inventivemethods and compositions to a specific autoimmune disorder illustratedby SLE. Details of experimental protocol are similar to those providedin Example 1.

CD3+ T or CD4+T_(M) cells were isolated from whole blood of eitherhealthy donors or SLE patients by negative selection using EasySepMagnetic Separation kit. As illustrated schematically in FIG. 5 and FIG.6, unilamellar lipid vesicles (100 nm diameter) composed ofL-α-phosphatidylcholine (PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (PE-PEG-biotin) and cholesterol (CH) were prepared andfunctionalized with streptavidin and biotinylated anti-CD45RO antibody.SiRNAs directed towards Kv1.3 channels or scrambled sequence RNA werethen incorporated in the vesicles. The donor cells were incubated withthe NPs overnight, activated with TG, and then subjected to antibodystaining and flow cytometry as described above.

Results.

i) KV1.3-NPs decrease NFAT nuclear translocation in T_(M).

It is known that T cell activation increases CD40L levels. FIG. 7 setsforth results of a flow cytometry analysis of CD40L expression in CD3⁺ Tcells that were resting or activated for 3 h with eitheranti-CD3/anti-CD28 antibodies, TG or PMA/lonomycin. CD3 cells weretreated with fluorescently labeled Kv1.3-NPs or NPs loaded with scrambleRNA (scr-NP) for 24 h. Ca²⁺ influx was induced with thapsigargin (TG,which facilitates the release of Ca²⁺ from intracellular stores andopens Ca²⁺ channels) for 1 h. Resting T cells (no TG treatment)transduced with scr-NPs were used as controls. Cells were fixed andstained with anti-NFAT antibody and DAPI (nuclear stain). Images wereacquired on the Image Stream imaging flow cytometer (Amnis, EMDMillipore). Nuclear translocation of NFAT was quantitated using theIDEAS software (Amnis, EMD Millipore) by gating on cells that hadincorporated the NPs. FIGS. 8A, 8B and 8C set forth representativeimages of cells acquired by imaging flow cytometry showing nucleartranslocation of NFAT in activated CD3 T cells and the absence ofnuclear translocation in resting T cells. In particular, FIG. 8A showsrepresentative flow cytometry images for activated T cells aftertreatment with scr-NPs and (FIG. 8B) siKv1.3-NPs. and FIG. 8C showsrepresentative images for resting T cells treated with null-NPs andstained with NFAT (green-gray scale) and DAP1 (yellow-gray scale). Thenuclear translocation of NFAT is indicated by the colocalization of redand green channels in the merged images. FIG. 9 shows the percentagenuclear translocation of NFAT in activated T cells treated with scr-NPsor Kv1.3-NPs, or resting T cells treated with scr-NPs, and FIG. 10 showsCD40L expression in CD45RO⁺ activated T cells from 3 healthy donorstransduced with NPs. Data show mean±SEM for 3 independent healthydonors, with 2300-3500 cells recorded per donor. Data are normalized toactivated null-NPs. *p<0.05.

ii) KV1.3-NPs decrease CD40L expression in T_(M) cells from SLEpatients.

FIGS. 11 and 12 set forth results from the following protocol: CD3 cellswere isolated from healthy donors and treated with either fluorescentlylabeled Kv1.3-NPs or scr-NPs or NPs without siRNA (null) for 24 h. Ca²⁺influx was induced by TG for 3 h. Resting T cells (No TG treatment, nointracellular Ca²⁺ influx) transduced with scr-NPs were used ascontrols. Cells were stained with anti-CD40L antibody and analyzed byflow cytometry. Only cells expressing the fluorescent NPs were gated foranalysis. FIG. 13 is a graphical representations showing CD40Lexpression in activated cells treated with Kv1.3-NPs, scr-NPs andnull-NPs and resting cells containing scr-NPs. Data are normalized tonull-NPs. Data show mean±SEM for 3 independent healthy donors, with50,000 total cells recorded in each experiment.

FIG. 14 sets forth a flow cytometry analysis of CD45RO and CD40Lexpression in CD3⁺ T cells from an SLE patient treated with siKv1.3-NPs,scr-NPs and neither (null-NPs). NPs were coated with Alea488-anti-CD45ROantibodies and activated with TG. The resting T cells with null-NPs wereused as controls. Average CD40L expression in CD45RO⁺ orCD45RO⁻-activated T cells from 3 SLE patients transduced with NPs areset forth graphically in FIGS. 15A and 15B. Data are normalized toactivated null-NPs. FIG. 16 is a histogram of CD40L expression inactivated CD45RO⁺ CD3⁺ cells from one SLE patient treated with eitherscr-NP or siKv1.3-NPs. FIG. 17 represents the average mean fluorescenceintensity (MFI) of CD40L expression in activated CD45RO⁺ CD3⁺ cells fromSLE patients transduced with scr-NPs or siKv1.3-NPs (data normalized toMFI of scr-NPs).

iii) Kv1.3-NPs induce loss of CD45RO expression in SLE T cells

FIG. 18 sets forth a flow cytometry analysis for CD45RO expression inCD3⁺ cells isolated from one SLE patient incubated with null-, scr- orsiKv1.3-NPs and then activated with TG. As depicted, T_(M) (CD45RO⁺) andCD45RO⁻ populations were identified by drawing rectangle gates. FIG. 19is a graphical representation of the ratio CD45RO⁺/CD45RO⁻ in activatedCD3 cells isolated from three SLE patients.

iv) Kv1.3-NPs decrease CD45RO expression in CD4⁺T_(M) cells and switchthe cell phenotype from CD45RA⁻ to CD45RA⁺

FIG. 20 sets forth results of flow cytometry experiments for CD45RO andCD45RA expression in CD4⁺T_(M) cells isolated from a healthy donor andincubated with either scr- or siKv1.3-NPs and then activated with TG.FIG. 21 shows viability for cells shown in FIG. 20 measured by flowcytometry using the nuclear dye 7-AAD.

The foregoing example demonstrates that (1) Kv1.3-NPs selectively targetT_(M) cells from healthy donors resulting in decreased Ca²⁺ influx,inhibition of NFAT nuclear translocation, decreased CD40L expression,and induction of memory loss, i.e. reversion of CD45RO⁺/CD45RA⁻ cells toCD45RO⁻/CD45RA⁺, and (2) Kv1.3-NPs selectively target T_(M) cells fromSLE patient donors resulting in decreased CD40L expression and decreasedCD45RO expression.

The present experiments as well as ongoing empirical studies by theinvestigators support therapeutic efficacy in the treatment ofautoimmune disorders characterized by hyperactive or over expression ofT_(M) cells, exemplified by SLE. The demonstrated reduction in Ca²⁺influx results in a decrease in T_(M) hyperactivity and a decrease inCa²⁺ dependent CD40L expression in T_(M) cells, which prevents B cellactivation and autoantibody production. The demonstrated selectivity fora specific T cell subset overcomes the known problem of systemicinhibition associated with administration of Kv1.3 channel blockers.

It is expressly contemplated that each of the various aspects,embodiments, and features thereof described herein may be freelycombined with any or all other aspects, embodiments, and features. Theresulting aspects and embodiments (e.g., compositions and methods) arewithin the scope of the invention. It should be understood that headingsherein are provided for purposes of convenience and do not imply anylimitation on content included below such heading or the use of suchcontent in combination with content included below other headings.

All articles, books, patent applications, patents, other publicationsmentioned in this application are incorporated herein by reference. Inthe event of a conflict between the specification and any of theincorporated references the specification (including any amendmentsthereto) shall control. Unless otherwise indicated, art-acceptedmeanings of terms and abbreviations are used herein.

In the claims articles such as “a”, “an” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. It isto be understood that the invention encompasses all variations,combinations, and permutations in which one or more limitations,elements, clauses, descriptive terms, etc., from one or more of thelisted claims is introduced into another claim. For example, any claimthat is dependent on another claim may be modified to include one ormore elements, limitations, clauses, or descriptive terms, found in anyother claim that is dependent on the same base claim. Furthermore, wherethe claims recite a composition, it is to be understood that methods ofusing the composition according to any of the methods disclosed herein,and methods of making the composition, are included within the scope ofthe invention, unless otherwise indicated or unless it would be evidentto one of ordinary skill in the art that a contradiction orinconsistency would arise.

Where elements are presented as lists, it is to be understood that eachsubgroup of the elements is also disclosed, and any element(s) may beremoved from the group. The invention provides all such embodiments.

The terms “approximately” or “about” in reference to a number generallyinclude numbers that fall within ±10%, in some embodiments ±5%, in someembodiments ±1%, in some embodiments, ±0.5% in some embodiments, of thenumber unless otherwise stated or otherwise evident from the context(except where such number would impermissibly exceed 100% of a possiblevalue). Where ranges are given, endpoints are included. Furthermore, itis to be understood that unless otherwise indicated or otherwise evidentfrom the context and understanding of one of ordinary skill in the art,values that are expressed as ranges may assume any specific value orsubrange within the stated ranges in different embodiments of theinvention, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise. Any one or moreembodiment(s), element(s), feature(s), aspect(s), component(s) etc., ofthe present invention may be explicitly excluded from any one or more ofthe claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described and exemplified herein. The scopeof the present invention is not intended to be limited to the aboveDetailed Description and Examples, but rather is as set forth in theappended claims.

What is claimed:
 1. A method of treating a patient suffering from acondition of the immune system characterized by overexpressed and/orhyperexcitable T-cells, the method comprising administering to thepatient a composition formulated to selectively bind to aCD45RO-positive isoform of the T-cells and transfect it with siRNA, thecomposition comprising: lipid nanovesicles functionalized withsurface-bound antibody selective for a membrane protein unique to theCD45RO-positive isoform; and siRNA effective for inhibiting expressionof a Kv1.3 ion channel of the CD45RO-positive isoform cells upontransfection, said siRNA encapsulated within the lipid nanovesicle andcomprising one or more Kv1.3-specific siRNA.
 2. The method according toclaim 1, wherein the antibody comprises anti-CD45LRO.
 3. The methodaccording to claim 1, wherein the T-cell comprises a T_(M) cell.
 4. Themethod according to claim 2, wherein the antibody comprises monoclonalantibody.
 5. The method according to claim 1, wherein the siRNA isselected from the group consisting of GUAACUCGACUCUGAGUAAtt (SEQ ID NO:1), UUACUCAGAGUCGAGUUACtt (SEQ ID NO: 2), CUUACCCUCUCUCUCUUAAtt (SEQ IDNO: 3), UUAAGAGAGAGAGGGUAAGtt (SEQ ID NO: 4), GAUGGACCUUUCAACGUUAtt (SEQID NO: 5), UAACGUUGAAAGGUCCAUCtt (SEQ ID NO: 6), and duplexes thereof.6. The method according to claim 1, wherein administering is via asystemic route.
 7. The method according to claim 6, whereinadministering is via intravenous administration.
 8. The method accordingto claim 1, wherein the condition of the immune system comprises anautoimmune disorder selected from Rheumatoid arthritis, Type 1 diabetes,Multiple Sclerosis, Psoriasis, Vasculitis, Alopecia areata, SystemicLupus Erythematosus, Polymyalgia rheumatica, Ankylosing spondylitis,Celiac disease, Sjögren's syndrome, and Temporal arteritis.
 9. Themethod according to claim 8, wherein the condition of the immune systemis Systemic Lupus Erythematosus.
 10. The method according to claim 1,wherein the step of administering comprises dosing according to aschedule based on severity of the disease across a therapeutic timeframe.
 11. The method according to claim 10, wherein the severity of thedisease is assessed by calculating a ratio of T_(M) to nave T cells inplasma of the patient sampled across the therapeutic time frame.
 12. Amethod of treating a patient suffering from chronic systemic lupuserythematosus, the method comprising: administering to the patient acomposition comprising lipid nanovesicles surface-functionalized withanti-CD45LRO and encapsulating Kv1.3-specific siRNA.